Chloride is the most abundant anion in extracellular fluids and plays critical roles in numerous essential physiological processes including epithelial fluid transport, cell volume control, acid-base homeostasis and regulation of cell excitability. Anion channels mediate CI- transport across cell and organelle membranes in all organisms. Electrophysiological studies have identified a diverse array of anion channel types, but relatively little is known about anion channel molecular biology and regulation. The nematode C. elegans provides numerous experimental advantages for defining anion channel and CI- transport molecular physiology. A drawback of C. elegans, however, is its small size and limited physiological access. We recently developed a number of innovative methods that allow us to circumvent these problems. Using electrophysiology and reverse genetics, we identified a CIC CI- channel, CLH-3b, that is expressed in the C. elegans oocyte. CICs function in organisms from bacteria to animals and their physiological importance is underscored by the identification of mutations in five human CIC genes that give rise to kidney, muscle, bone and neurological disorders. The biophysical properties of CLH-3b resemble those of mammalian CIC-2. CLH-3b is activated by dephosphorylation during oocyte meiotic maturation and swelling, and functions to couple cell cycle progression to ovulation. The type 1 phosphatases CeGLC-7a/p and a newly identified Ste20 kinase, GCK-3, regulate CLH-3b. GCK-3 binds to CLH-3b, inactivates the channel in a phosphorylation dependent manner, and is a homolog of mammalian PASK. PASK regulates Na-K-2CI cotransporters involved in fluid secretion, osmoregulation, and cell volume and CI- homeostasis. This renewal application builds on the considerable progress and successes of the previous funding cycle of DK61168. During the next funding period we will use a combination of phosphopeptide analysis, forward and reverse genetics, electrophysiology, molecular biology and microscopy to 1) identify CLH-3b regulatory phosphorylation sites, 2) define the physiological roles of the CLH-3b regulatory kinase GCK-3, 3) identify components of the GCK-3 signaling cascade that regulates CLH-3b and whole animal fluid balance, and 4) begin characterizing the mechanisms and genes involved in regulating CLH-3b plasma membrane retrieval. Our proposed studies will provide significant new insights into CIC regulation, the function of GCK- 3 and its mammalian homolog PASK, and fundamental processes such as cell volume sensing and the coordinated regulation of ion channels and transporters that control cellular CI- content, epithelial fluid transport and cell volume. Detailed understanding of CIC regulation and GCK-3/PASK signaling is essential in order to define the role of anion channels in disease processes and their potential as therapeutic targets as well as to fully understand and treat fluid secretory diseases. [unreadable] [unreadable]